| ABSTRACT: Yellowtail flounder, Limanda
ferruginea, were collected from six locations along coastal New
England (i.e., Cape Cod
Bay, Georges Bank, Massachusetts Bay, mouth of the Merrimack River,
Mud Patch (south of Marthas Vineyard), and New
York Bight), and were monitored for blood chemistry and hematology. Plasma
osmolality, sodium, potassium, calcium,
hemoglobin, hematocrit, and mean corpuscular hemoglobin concentration
(MCHC) were measured.
Osmolality and sodium concentrations were frequently reduced during
spring. Potassium was generally elevated during
spring and/or summer, and reduced during fall and/or winter. Calcium
was usually highest during fall. The hematological
indices of hematocrit, hemoglobin, and MCHC showed an overall elevation
during winter as compared to fall. Fish from the
inshore locations of Cape Cod Bay, Massachusetts Bay, Merrimack River,
Mud Patch, and New York Bight differed
significantly from fish from the offshore reference location of Georges
Bank for particular blood parameters during certain
seasons. These data provide a baseline range for
blood constituents of yellowtail flounder over an annual cycle and at key
locations in the Northeast U.S.
Shelf Ecosystem. These results present evidence of possible anthropogenic
effects on the blood chemistry and hematology of yellowtail flounder
collected from inshore areas. Variability in blood chemistry appears
to be regulated by seasonally-induced physiological and/or environmental
factors, and influenced by sex and capture location. This information
may prove to be useful in monitoring the health of field-collected yellowtail
flounder and in assessing the condition of aquacultured fish of this
species.
INTRODUCTION
Yellowtail flounder, Limanda ferruginea, is a commercially important
flatfish inhabiting Northwest Atlantic waters ranging from the Gulf of
St. Lawrence to Chesapeake Bay (Bigelow and Schroeder 1953; Collette
and Klein-MacPhee 2002). Evidence suggests that yellowtail flounder from
Cape Cod Bay, Georges Bank, and the region encompassing Southern New
England to the Mid-Atlantic Bight represent three distinct stocks (Cadrin
2003; Cadrin and King 2003). Yellowtail flounder are fast growing, have
high market value (Johnson et al. 1999), and have been identified
as a candidate species for aquaculture (Rabe and Brown 2000). Despite
the historical importance of yellowtail flounder as a commercial resource,
relatively little information is available concerning the blood chemistry
of this species.
Blood chemistry and hematological measurements can provide
valuable tools for monitoring the health and condition of both wild and
cultured fish. Physiological indices can offer critical feedback on rearing
conditions and nutritional status, and can aid in the diagnosis of disease.
These monitoring and feedback applications require an understanding of
normal blood component levels specific to yellowtail flounder, as blood
chemistry concentrations are known to vary among fish species (Larsson et
al. 1976; Hardig and Hoglund 1983; Folmar 1993). Natural changes
in environmental conditions associated with season can affect blood chemistry
and hematology (Bridges et al. 1976; Warner and Williams 1977;
Lane 1979; Dawson 1990; Folmar 1993; Houston 1997; Luskova 1998; O'Neill et
al. 1998; Edsall 1999). Physiological variation in fish may be influenced
by both internal and external cues, including reproductive stage, water
temperature, dissolved oxygen, nutrient availability, and photoperiod,
all of which undergo annual cycles (Sandstrom 1989; Dawson 1990). Seasonal
changes may also be influenced by nonenvironmental factors such as diet,
metabolic adaptations, and activity levels (Denton and Yousef 1975).
Hematological measures, which can be affected by exposure
to chemical pollutants (Heath 1995), are useful indicators of sublethal
environmental stress in fish (Bridges et al. 1976; Warner and
Williams 1977; Folmar 1993). Yellowtail flounder demonstrate strong site
fidelity, are found both inshore and offshore (Royce et al. 1959;
Lux 1964; Johnson et al. 1999; Cadrin 2003; Cadrin and King 2003),
and live on or close to bottom sediments where contaminant loading occurs.
Resident bottom-dwelling organisms, such as flatfish, often exhibit environmentally-induced
disease (Ziskowski et al. 1987), making them potential indicator
species for monitoring of anthropogenic effects. Before blood constituent
data can be applied as a diagnostic tool, however, general patterns related
to season, sex, and collection location need to be well documented and
understood (Bridges et al. 1976; Courtois 1976; Hardig and Hoglund
1983; Folmar 1993).
The goal of this study was to determine baseline levels of blood
parameters for yellowtail flounder. Blood chemistry and hematological
measurements of osmolality, potassium, sodium, calcium, hematocrit, hemoglobin,
and mean corpuscular hemoglobin (MCHC) were determined for fish collected
from various locations in the Northeast U.S. Shelf Ecosystem. Blood measurements
were compared with regard to fish sex, season, and collection location.
MATERIALS AND METHODS
Yellowtail flounder were collected from Northwest Atlantic
waters between 1978 and 1985 aboard National Marine Fisheries Service,
Northeast Fisheries Science Center (NEFSC) cruises. Nearly all fish were
collected on cruises associated with the NEFSC's Ocean Pulse Program
(OPP) and Northeast Monitoring Program (NEMP); a few fish were collected
on cruises associated with the NEFSC's Bottom Trawl Survey Program. (The
OPP and NEMP were the same fundamental program; the name changed from
the former to the latter early in the effort, though.) The focus of NEMP
was assessment of ecological, genetic, pathological, and physiological
changes in coastal and shelf organisms of the Northeast U.S. Shelf Ecosystem,
which had been potentially exposed to the effects of contaminant stress
(Pearce 1998).
Yellowtail flounder were collected using a "¾ Yankee" (or "No.
36") otter trawl towed for a 30-min period (Survey Working Group,
Northeast Fisheries Center 1988). Animals were obtained from five nearshore
locations, including Cape Cod Bay, Massachusetts Bay, mouth of the Merrimack
River, Mud Patch (south of Marthas Vineyard on the continental shelf),
and New York Bight, as well as from an offshore location, Georges Bank
(Figure
1). Table 1 shows the general coordinates (i.e., latitude
and longitude) of the collection stations, as well as the number of adult
yellowtail flounder collected at each station. For the Georges Bank,
Mud Patch, and New York Bight locations, fish caught at adjacent stations
within a given location were pooled to increase sample size for statistical
analysis. Environmental conditions (e.g., depth, substrate, temperature)
at these adjacent stations were fundamentally similar.
Fish were transferred to flowing seawater aboard ship
and processed immediately to minimize stress-induced changes associated
with capture (Larsson et al. 1976; Wedemeyer and Yasutake 1977;
Dawson 1990). Changes in blood concentrations can occur within hours
to days of collection (Umminger 1970; Wedemeyer and Yasutake 1977; Bourne
1986); therefore, it was critical that samples collected at sea be processed
without delay. Blood was collected by cardiac puncture using a 3-ml plastic
syringe with a 22-ga needle and transferred gently into an 8-ml glass
vial coated with 150 units of dried ammonium heparin as an anticoagulant.
Hemoglobin (g/100 ml) was determined by the cyanmethemoglobin method
using Hycel reagents and a Bausch and Lomb Spectronic 20 spectrophotometer.
(Note: Use of trade names does not imply endorsement by NMFS.) Hematocrit
was determined
by collecting blood in micro-hematocrit tubes, which were then centrifuged
at 13,500 × g for 5 min and read. Hematrocrit was expressed
as a percentage of the volume of the whole blood sample. The remainder
of each blood sample was centrifuged at 12,000 × g, and the
resulting plasma was frozen until further analyses could be conducted.
The MCHC, the ratio between hemoglobin and hematocrit, was expressed
as g/100 ml of packed red blood cells, and was calculated by the formula:
MCHC = hemoglobin/hematocrit × 100. Plasma osmolality was determined
using an Advanced 3L or 3C2 Cryomatic Osmometer and Advanced Instruments
freezing-point calibration standards. Sodium, potassium, and calcium
(mEq/L) were determined using a Perkin Elmer Coleman 51 Flame Photometer.
Sampling dates were assigned to a season according
to calendar designations: winter (December 22 - March 19), spring (March
20 - June 20), summer (June 21 - September 22), and fall (September 23
- December 21). Some locations and seasons were sampled more frequently
than others because of problems often associated with extensive field
sampling at sea, such as equipment failure, inclement weather, and variation
in fish availability. Occasionally, a sample was not obtained for a given
season. Small sample sizes were included in a data set when information
was available for other seasons at that location.
A Pearson product moment correlation was used to determine
whether hydrographic characteristics such as temperature, salinity, and
dissolved oxygen are associated with blood chemistry values. A multivariate
analysis of variance was conducted to determine effects of sex, location,
and season on osmolality, sodium, potassium, calcium, hemoglobin, hematocrit,
and MCHC. These seven blood parameters served as the dependent variables.
Independent variables for the model included sex (males and females),
location (Cape Cod Bay, Georges Bank, Massachusetts Bay, Merrimack River,
Mud Patch, and New York Bight), season (winter, spring, summer, and fall),
sex × location, sex × season, and sex × location × season.
Fish length was used as a covariate. Post-hoc tests consisted of multiple
pairwise comparisons of least square means for significant effects (P<0.05).
Statistical comparisons were limited to seasons and locations containing
a sample size of N>5. The significant differences which have
been reported, but based on small sample sizes, should be interpreted
conservatively. All statistical analyses were performed using PC-SAS
Software Version 8.3 (SAS Institute 1989).
RESULTS
Two of the three hydrographic parameters correlated
highly with blood indices. Salinity correlated positively with MCHC (Table 2). Dissolved oxygen correlated positively with hemoglobin and MCHC,
and negatively with osmolality and sodium. There were also relationships
between certain blood parameters. Sodium correlated
positively with osmolality, and negatively with MCHC.
Hemoglobin correlated positively with hematocrit and MCHC.
A significant difference was observed for sex × location × season for
all seven blood parameters (Table 3). Following are results for differences related
to sex, season, and location. Location-related differences are limited to a comparison
of results from the five inshore locations versus those from Georges Bank, the
offshore reference location.
SEX-RELATED DIFFERENCES IN BLOOD CHEMISTRY
Significant differences in blood parameters were observed
between males and females within the same season at Georges Bank (8 instances),
New York Bight (5), Mud Patch (5), and Merrimack River (2), and are shown
underlined in Tables 4-10. These differences were observed most frequently
during spring (8), but were also observed during summer (6), winter (4),
and fall (2). Male and female blood chemistry concentrations differed
significantly from one another for hemoglobin (6), osmolality (4), hematocrit
(4), calcium (3), potassium (2), and MCHC (1).
Mud Patch males had significantly greater hematocrit
during winter, and hematocrit, hemoglobin, and osmolality during spring,
than females. Mud Patch females had significantly greater hemoglobin
during summer than males. Merrimack River females had significantly greater
calcium during spring and summer than males. New York Bight males had
significantly greater hematocrit and osmolality during winter, hemoglobin
and osmolality during spring, and potassium during fall, than females.
Georges Bank males had significantly greater hemoglobin during winter,
hematocrit and hemoglobin during spring, hemoglobin, potassium, and MCHC
during summer, and osmolality during fall, than females; however, females
had significantly greater calcium during summer than males.
SEASONAL DIFFERENCES IN BLOOD CHEMISTRY
Overall, osmolality showed few significant differences
among seasons, although there was a minor trend toward reduced osmolality
during spring, as observed in both males and females from Georges Bank
(Table 4). Fall osmolality in Mud Patch fish was significantly greater
than summer osmolality in males, and spring osmolality in females. Osmolality
in New York Bight males was significantly elevated during winter and
spring, and reduced during fall.
Generally, sodium concentrations were significantly
lower during spring, as observed in males from Georges Bank, Merrimack
River, Mud Patch, and New York Bight, and in females from Georges Bank
and Mud Patch (Table 5).
Potassium concentrations were often significantly higher
during spring or summer, and significantly lower during fall or winter
(Table 6). Potassium showed significantly higher levels during spring
in males from Merrimack River, and in females from Georges Bank and Mud
Patch.
Summer levels were significantly higher in fish of both
sexes from Georges Bank and Mud Patch. Potassium was
significantly lower during fall than spring and/or summer in
males from Georges Bank, Merrimack River, and Mud Patch,
and in females from Georges Bank. Winter potassium was
significantly lower than summer for females from Georges
Bank and Mud Patch.
Calcium concentrations were generally higher during
fall, as observed in fish of both sexes from George Bank and Mud Patch,
and in females from New York Bight (Table 7). A highly elevated calcium
concentration during spring was documented in females from Merrimack
River.
Yellowtail flounder demonstrated a trend toward significantly
higher hematocrit during spring, as compared with fall (Table 8). Hematocrit
was significantly greater during spring than fall in fish of both sexes
from Merrimack River, Mud Patch, and New York Bight, and in males from
Georges Bank. Winter hematocrit concentration was significantly greater
than fall concentration in females from Georges Bank.
Hemoglobin showed a similar pattern to that observed
for hematocrit, but the results were less consistent (Table 9). Hemoglobin
tended toward significantly greater concentrations during winter and/or
spring than fall. Hemoglobin in fish of both sexes from Georges Bank
and Merrimack River was significantly higher during winter than fall,
while spring levels were significantly higher than fall levels in males
and females from Merrimack River, Mud Patch, and New York Bight, and
in males from Georges Bank.
MCHC was generally higher during winter than spring,
as observed at Georges Bank and Merrimack River for males, and at Merrimack
River and New York Bight for females (Table 10). Winter also tended to
have higher MCHC than fall, as seen at Georges Bank and Merrimack River
for males, and at Merrimack River and Mud Patch for females.
LOCATION-RELATED DIFFERENCES IN BLOOD CHEMISTRY AS COMPARED TO GEORGES
BANK
Blood parameters in fish from the inshore locations
were compared to such parameters in fish from Georges Bank, the reference
location. During winter, osmolality in female yellowtail flounder from
Merrimack River was significantly lower. During spring, osmolality in
both sexes from New York Bight and in males from Cape Cod Bay was significantly
higher. During summer, osmolality in both sexes from Merrimack River
and in males from Mud Patch was significantly lower. During fall, osmolality
in males from Merrimack River, Mud Patch, and New York Bight was significantly
lower (Table 4).
During winter, Merrimack River, Mud Patch, and New
York Bight sodium concentrations in females were significantly reduced.
During spring, sodium in females from Massachusetts Bay, males from Cape
Cod Bay, and both sexes from New York Bight was significantly elevated.
Fall sodium concentrations were reduced in both sexes
from Merrimack River (Table 5).
Reduced potassium was observed during spring in males
from Cape Cod Bay and in females from Massachusetts Bay and New York
Bight. Merrimack River showed elevated potassium during spring in males.
Significantly reduced potassium was measured in Merrimack River males
during summer. Females from Merrimack River and Mud Patch and both sexes
from New York Bight had elevated fall potassium (Table 6).
Females from Merrimack River and Massachusetts Bay
had significantly higher calcium during spring. Summer calcium was reduced
in female fish from Mud Patch. Calcium concentrations during fall were
significantly lower in both sexes from Merrimack River and in males only
from Mud Patch (Table 7).
During winter, hematocrit was significantly lower in
males from Cape Cod Bay and in females from Mud Patch. During spring,
hematocrit was significantly higher in both sexes from Merrimack River,
and in females from New York Bight. During summer, hematocrit was significantly
higher in females from New York Bight (Table 8).
During winter, hemoglobin in males from Cape Cod Bay
and New York Bight, and in both sexes from Mud Patch, was significantly
reduced. Merrimack River females had greater winter hemoglobin. During
spring, hemoglobin was reduced in female fish from Massachusetts Bay
and elevated in females from Merrimack River. Summer hemoglobin was reduced
in Mud Patch males. Fall hemoglobin in New York Bight and Mud Patch males
was significantly higher (Table 9).
Males from Cape Cod Bay and New York Bight had significantly
lower winter MCHC, while in females from Merrimack River, MCHC was significantly
greater. During summer, males from Merrimack River and females from New
York Bight had significantly reduced MCHC. Male fish from Merrimack River,
Mud Patch, and New York Bight had significantly higher MCHC during fall
(Table 10).
DISCUSSION
SEASONAL AND SEX-RELATED DIFFERENCES IN BLOOD CHEMISTRY
Blood chemistry and hematological measurements are
significantly affected by the wide range of natural environmental conditions
experienced by fish over the course of a year (Bridges et al. 1976;
Warner and Williams 1977; Lane 1979; Dawson 1990; Folmar 1993; Houston
1997; Luskova 1998; O'Neill et al. 1998; Edsall 1999). Changes
in temperature (Powers 1980) and season (Shell 1961) are known to affect
oxygen metabolism and hematology. Variations in blood constituents may
be related to natural physiological cycles, environmental stimuli, or
both (Bridges et al. 1976; Luskova 1998). Metabolic fluctuations
in fish such as growth, activity, and feeding are strongly influenced
by environmental cues (Barnhart 1969; Fletcher 1977).
Reproductive cycles, associated with season, can place fish
under extremes of metabolism and osmoregulation
(Courtois 1976), and may alter the content of fish blood (Nagler et al. 1987; Sandstrom 1989; Bjornsson et
al. 1998; Luskova 1998).
Osmolality
Yellowtail flounder experienced only minor seasonal
fluctuations. In several cases, osmolality values were reduced during
spring. Windowpane (Scophthalmus aquosus) also experience significantly
lower osmolality during spring than summer and fall (Dawson 1990). In
a laboratory study, however, no significant differences were observed
in the serum osmolality of winter flounder (Pseudopleuronectes americanus)
held at 1° and 15°C (Umminger 1970).
The significantly elevated osmolality observed during
winter and spring in males from New York Bight in our study has some
adaptive significance. Pearcy (1961) suggests that elevated osmotic pressure
in flounder during cooler weather may protect against freezing in fishes
inhabiting cold shallow waters. Serum osmolality was also highest in
winter flounder during the winter months, just prior to spawning (Pearcy
1961; Umminger and Mahoney 1972). Osmolality in mummichog (Fundulus
heteroclitus) was found to increase significantly at colder water
temperatures (Umminger 1969).
Seasonal differences in osmotic pressure may be caused
by osmotic imbalance or other factors (Pearcy 1961). An increase in serum
constituents other than the major ions, such as a buildup of organic
and inorganic materials, can elevate osmolality during cold weather (Pearcy
1961; Umminger 1969; Umminger and Mahoney 1972). An increase in serum
electrolytes may be a compensatory mechanism used by fish to maintain
the total number of osmotically active particles in the plasma (Shell
1961). In several instances, males showed significantly greater osmolality
than females during winter and/or spring. Perhaps females are undergoing
osmotic changes related to preparations for
late spring spawning.
In our study, osmolality and sodium were highly positively
correlated. This high positive correlation would be expected, as sodium
is an important osmotic constituent. In our study, both of these parameters
had similar patterns of reductions during spring. Also in our study,
dissolved oxygen and osmolality were highly negatively correlated, which
suggests that the reduced osmolality during spring may be associated
with the elevated dissolved oxygen levels following spring turnover.
Sodium
Marine fish exposed to low seawater temperatures often
experience increased sodium (Umminger 1969; Murphy and Houston 1977;
Sandstrom 1989). Increases in freezing point depression of winter flounder
blood serum during the winter months can be attributed in part to elevated
plasma sodium (Fletcher 1981). Winter flounder sampled in
Newfoundland had significantly higher sodium during
winter than summer (Fletcher 1977), while sodium in winter
flounder from New England was also higher during late
winter (Pearcy 1961; Fletcher 1977). Winter-collected
windowpane had significantly higher sodium than summer and
autumn fish (Dawson 1990). Interestingly, Umminger and
Mahoney (1972) noted no seasonal change in sodium among
winter flounder collected from New Jersey.
In measurements on freshwater fish, sodium levels in
the Eurasian perch (Perca fluviatilis) reached their maximum during
mid-winter (Sandstrom 1989), and in rainbow trout (Oncorhynchus mykiss)
were higher during both fall and winter than during summer (Houston et
al. 1968). In our study, dissolved oxygen concentrations were correlated
negatively with both sodium and osmolality. This correlation suggests
a possible disruption of osmoregulatory function at reduced oxygen levels.
Potassium
The significantly reduced potassium levels during cold
weather, which were observed in our study, have also been described in
winter flounder, and may be related to a decline in metabolism and feeding
at lower temperatures (Umminger and Mahoney 1972; Bentinck-Smith et
al. 1987). Reduced blood potassium concentrations during winter may
also be attributed to a loss of potassium from the cells of body tissues
in response to the cold (Umminger 1969). Potassium in the windowpane
was also found to be higher during summer than fall and winter (Dawson
1990).
Changes in blood potassium levels have also been observed
in freshwater fish. Rainbow trout collected during fall-winter had reduced
levels as compared to those fish collected during summer (Houston et
al. 1968). Elevated potassium levels were also observed at high water
temperatures in smallmouth bass (Micropterus dolomieu) (Shell
1961), rainbow trout (Murphy and Houston 1977), channel catfish (Ictalurus
punctatus) (Ellsaesser and Clem 1987), Eurasian perch (Sandstrom
1989), and madai (Pagrus major) (Woo 1990).
Increased plasma potassium may result from disruption
of potassium regulatory ability at either the external level (i.e.,
between the fish and seawater) or internal level (i.e., between
the intracellular and extracellular fluid), with a resulting release
of potassium into the blood. Serum potassium levels may increase in response
to elevated tissue catabolism, or due to osmotic adjustment when compensating
for a decline in other serum components (Shell 1961).
Calcium
Calcium plays an important role in osmoregulation (Shell
1961), and is a critical component in the reproductive processes of fish.
In our study, calcium concentrations were generally higher during fall
and lower during spring.
This trend toward calcium being elevated during fall and
depressed during spring may reflect the spring and
summer spawning pattern of yellowtail flounder, which peaks
during late May (Bigelow and Schroeder 1953; Royce et al. 1959; Fahay 1983; Collette and Klein-MacPhee 2002; Cadrin
and King 2003). Also in our study, a contrasting pattern was observed
in females from Merrimack River; they showed significantly elevated calcium
during spring which may be related to anthropogenic, rather than seasonal,
influences.
In our study, female fish from Georges Bank and Merrimack
River had significantly elevated spring and/or summer calcium levels
as compared to males. These elevated calcium levels in females are most
likely related to hormonal changes linked to spawning activity. In freshwater
fish also, calcium concentrations in females during the spawning period
were significantly higher than in males for lake trout (Salvelinus
namaycush)(Edsall 1999), brook trout (Salvelinus fontinalis)(Booke
1964), and rock bass (Ambloplites rupestris)(Bidwell and Heath
1993).
Blood calcium has been shown to follow spawning patterns
in numerous fish including brook trout (Booke 1964), rainbow trout (Nagler et
al. 1987), English sole (Parophrys vetula)(Johnson et al. 1991),
striped mullet (Mugil cephalus), pinfish (Lagodon rhomboides)
(Folmar et al. 1992), rock bass (Bidwell and Heath 1993), and
Atlantic halibut (Hippoglossus hippoglossus) (Bjornsson et
al. 1998). Blood calcium in rainbow trout rises in connection with
yolk synthesis and reaches maximum levels just prior to spawning (Hille
1982). High spring calcium levels observed in windowpane (Dawson 1990)
appear to correspond with a late spring and summer spawning season (Bigelow
and Schroder 1953). Calcium varied irregularly in the Eurasian perch,
with fluctuations increasing sharply before and after spring spawning
(Sandstrom 1989). Variations in blood calcium of fish appear to be tied
more closely to reproductive changes than to seasonality or hematopoiesis
(i.e., blood formation) (Luskova 1998).
Hematocrit, Hemoglobin, and MCHC
Hematocrit and Hemoglobin
Hematocrit provides a measurement of red blood cells
(erythrocytes) in whole blood, while the hemoglobin within those erythrocytes
is the main transport mechanism for oxygen and carbon dioxide in the
blood. Alterations in blood oxygen capacity reflect seasonal adjustment
in oxygen transport (Cameron 1970; Anderson et al. 1985). Elevations
in spring hematocrit and hemoglobin are likely due, in part, to an increase
in oxygen consumption and metabolic rates corresponding to a rise in
water temperatures (Powers 1980; Dwyer et al. 1983; Zanuy and
Carrillo 1985; Martinez et al. 1994). Spring hemoconcentration
of the blood may also contribute to elevated hematological levels (Preston
1960).
In addition to these seasonal rhythms, sex also appears
to play a role in hematocrit and hemoglobin levels. In our study, males
sometimes showed higher hematocrit and hemoglobin levels than females
during winter and spring. Lane (1979) also observed significantly higher
hematocrit and hemoglobin levels in male versus female rainbow trout.
These differences in hematology with regard to males and females may
be related to differential oxygen demand by sex, which in turn may be
related to reproductive activity. Elevations in hematocrit and hemoglobin
of rock bass seemed to occur in relation to the onset of spawning (Bidwell
and Heath 1993).
The fall reduction in yellowtail flounder hematocrit
and hemoglobin levels observed in our study may reflect a corresponding
hemodilution of the blood which results in decreased hematological parameters
and/or a reduction in hemopoetic capacity (Preston 1960). Rhythms in
hematocrit and hemoglobin concentration may also result from changes
in plasma volume or erythrocyte volume (Sandstrom 1989), for example,
an expansion of plasma volume reduces the density of circulating red
blood cells, thereby decreasing hematocrit (Courtois 1976). Changes in
metabolism and hormonal activity, triggered by cooler water temperatures
and declining photoperiod during fall, may result in anemia (Lane 1979)
and reduced erythropoetic production (Lane 1979; Zanuy and Carrillo 1985).
Physiological stresses associated with fasting and spawning can trigger
a decline in fish condition (Bridges et al. 1976; Sano 1960ab;
Lane 1979; Zanuy and Carrillo 1985).
Seasonal hematological patterns vary among species.
In plaice, Pleuronectes platessa, hematocrit was lowest during
winter, increased through the spring into summer, and declined again
into fall. Hemoglobin levels in that species were high at the end of
winter, were lowest during spring, were again high during the summer,
and had a further decline during late fall (Preston 1960). In windowpane,
Dawson (1990) found significantly lower winter hematocrit. Winter flounder
from Maine had the lowest levels of hematocrit and hemoglobin during
winter and early spring (Bridges et al. 1976), while winter flounder
from New Jersey had lower levels of hemoglobin during late winter (Umminger
and Mahoney 1972). [ERRATUM (posted May 10, 2004): A
more comprehensive analysis of Hudson-Raritan Estuary winter flounder
hematological variables (Mahoney and McNulty 1992) showed hemoglobins
and hematocrit values to be high from October to April and low from June
through September; values in May were transitional.]
Tun and Houston (1986) found increased hematocrit
and hemoglobin in rainbow trout exposed to summer versus winter conditions.
An overall trend toward increased hemoglobin was observed in pinfish
and striped mullet held at high temperatures (Cameron 1970). Hematocrit
and hemoglobin in striped bass (Morone saxatilis) were highest
during fall and winter and lowest during summer (Lochmiller et al. 1989).
Haider (1969) found hemoglobin in rainbow trout to be highest during
winter and lowest during fall.
MCHC
Changes in the hemoglobin content of the blood in response
to the environment might come about either by a change in the number
of erythrocytes or by a change in the hemoglobin concentration of the
individual cells
(Anthony 1961). MCHC, the amount of hemoglobin in a given
number of red blood cells, was generally higher during winter
than spring and fall. Since MCHC values are directly related
to erythrocyte maturation, the relatively high proportion
of newly proliferated young erythrocytes during late spring
-- when fish are in a state of rapid growth -- is
associated with relatively low MCHC values (Denton and Yousef
1975; Hardig and Hoglund 1983). MCHC corresponded with
hematocrit and hemoglobin in striped bass, being
highest during fall and winter and lowest during summer
(Lochmiller et al. 1989). MCHC in the largescale blackfish
(Girella punctata), increased during spring and decreased during fall
(Kakuno and Koyama 1994). Dawson (1990) observed no seasonal or location differences
in MCHC among windowpane collected from various locations in Long
Island Sound.
[ERRATUM (posted May 10, 2004): Mean MCHC
in winter flounder from the Hudson-Raritan Estuary was generally highter during
the period October through April as compared to May through September (Mahoney
and
McNulty 1992).]
As hemoglobin is a measure of the amount of oxygen
carried in the blood, the strong positive correlation observed in our
study between dissolved oxygen and two hematological indices, hemoglobin
and MCHC, would be expected. Similarly, Kakuno and Koyama (1994) observed
a strong positive correlation between hematological parameters and
dissolved oxygen in teleost fishes. Also in our study, MCHC correlated
positively
with salinity and negatively with sodium concentration. These results
suggest that hematological indices may be affected by seasonal changes
in hydrographic conditions.
LOCATION-RELATED DIFFERENCES IN BLOOD CHEMISTRY AS COMPARED TO GEORGES
BANK
One of the most important potential uses of blood chemistry
data from a toxicological perspective is to assess environmental quality
by comparing animals from different locations (i.e., reference
versus contaminated locations) (Folmar 1993). Physiological biomonitoring,
using hematological and metabolic indices to assess stress, could provide
an alternative to traditional population-based approaches (Lochmiller et
al. 1989). A knowledge of normal physiological values is necessary
to evaluate the health of fish with respect to their physiological responses
to a stimulus or stress which affects homeostasis (Luskova 1998). A variety
of environmental pollutants, such as certain chlorinated hydrocarbons
and heavy metals, have been shown to affect osmotic and ionic regulation
in fish (Haux 1979; Heath 1995).
Differences in blood parameters between yellowtail
flounder from inshore locations and those from Georges Bank, the offshore
location, suggest a possible anthropogenic effect at several inshore
locations. Blood constituents that are significantly elevated or reduced
in inshore fish as compared to Georges Bank fish may indicate a physiologically
stressed condition (Reid et al. 1987). By virtue of location,
the Georges Bank location experiences minimal input from anthropogenic
sources, whereas the inshore locations are known to contain contaminated
sediments (Boehm 1983). In a summary of the NEMP, Reid et al. (1987) documented elevated trace metals in the sediments
from New York Bight, Mud Patch, and Massachusetts Bay, as well at the
mouths of coastal estuaries. New York Bight had the highest measurements
of polychlorinated biphenyls, while the incidence of fin rot at that
location was found to be significantly greater than at other Northwest
Atlantic locations (Ziskowski et al. 1987).
Osmolality and Sodium
Significantly lower osmolality levels in Merrimack
River, Mud Patch, and New York Bight male yellowtail flounder during
fall, and significantly lower sodium levels in Merrimack River males
and females during the same season, suggest reduced osmoregulatory capacity,
and may indicate an anthropogenic effect, as chronic low osmolality levels
are thought to result from exposure to contaminants (Wedemeyer and Yasutake
1977). Dawson (1990) documented significantly reduced osmolality in windowpane
from a polluted harbor in Long Island Sound as compared to fish from
other, less impacted areas. European flounder (Platichthys flesus)
exposed to titanium dioxide industrial effluent experienced reductions
in osmolality and blood sodium (Larsson et al. 1980). Reduced
blood sodium was documented in rainbow trout exposed to chlorine (Zeitoun et
al. 1977). Leakage of sodium and other ions from extracellular fluids,
or decreased uptake of these ions through the gills, can contribute to
impaired osmoregulatory ability (Pearcy 1961; Larsson et al. 1980).
The significantly elevated spring osmolality and blood
sodium levels observed in New York Bight male and female yellowtail flounder
may indicate a pollutant effect. Windowpane exposed to mercury in the
laboratory showed elevated sodium concentrations (Dawson 1990). A marked
increase in blood sodium was observed in seawater-acclimated rainbow
trout exposed to bunker C oil, most likely resulting from direct interference
of the oil with the sodium transport system of the gills (McKeown and
March 1978).
Potassium
Significantly elevated potassium levels were observed
during fall in yellowtail flounder males and females from New York Bight,
and in females from Merrimack River and Mud Patch. These elevated levels
may result from impaired uptake of potassium ions through the gills,
disruption of food absorption in the intestinal mucosa, or defective
renal function and impaired reabsorption of potassium ions in the renal
tubes (Larsson et al. 1981).
Elevated potassium is also known to result from chlorine
exposure in rainbow trout (Zeitoun et al. 1977). European flounder
exposed to sublethal cadmium concentrations showed a dose-dependent reduction
in
potassium concentration (Larsson et al. 1981).
Calcium
Calcium was significantly elevated in Massachusetts
Bay and Merrimack River females during spring, while levels were significantly
reduced in females from Mud Patch during summer, and in fish of both
sexes from Merrimack River and males from Mud Patch during fall. These
values differed significantly from Georges Bank, perhaps symptomatic
of impaired physiological processes. Larsson et al. (1981) speculates
that a disturbance in calcium metabolism of fish may be caused by defective
calcium absorption or impaired calcium reabsorption in the renal tubes,
or a combination of these effects.
In laboratory exposures, windowpane exposed to mercury
(Dawson 1990) and European flounder exposed to cadmium experienced reduced
calcium concentration (Larsson et al. 1981).
Hematocrit, Hemoglobin, and MCHC
With only one exception, yellowtail flounder from Cape
Cod Bay had the lowest levels of hematocrit, hemoglobin, and MCHC by
sex during winter. These levels for Cape Cod Bay fish were also among
the lowest levels over all seasons and locations in our study. Mud Patch
females had the lowest level of winter hematocrit; they also had the
next-to-lowest level of winter hemoglobin. Similarly, anemic summer flounder
(Paralichthys dentatus), winter flounder, and windowpane were
also collected from Cape Cod Bay and Mud Patch during NEMP cruises (Reid et
al. 1987). Reduced hematocrit and/or hemoglobin can indicate physiological
stresses such as anemia, dehydration, and hemodilution resulting from
either gill damage (Wedemeyer and Yasutake 1977) or increased red blood
cell breakdown in the spleen (Larsson et al. 1980). Reduced MCHC
suggests disruption of the hemoglobin-forming process as opposed to the
rate of blood cell production (Heath 1995). Reduced hematocrit, hemoglobin,
and MCHC were documented in redear sunfish (Lepomis microlophus)
collected next to a selenium discharge site; the selenium appeared to
interfere with normal hemoglobin formation, evidenced by the disproportionate
reduction in hemoglobin as compared to red blood cells (Sorensen and
Bauer 1983). European flounder exposed to cadmium in the laboratory experienced
significant reductions in hematocrit and hemoglobin with no associated
change in MCHC. In this case, the cadmium-induced anemic response may
be due to increased blood plasma volume, accelerated loss or destruction
of erythrocytes, and/or decreased rate of erythrocyte production (Johansson-Sjobeck
and Larsson 1978).
During spring, elevated hematocrit was noted in yellowtail
flounder males and females from Merrimack River, and females from New
York Bight; it likely represents a swelling of cells in response either
to high blood levels of carbon dioxide or to low pH rather than an increase
in the number of circulating erythrocytes (Heath 1995), as no significant
difference was noted in MCHC. Hematocrit may increase in response to
stress at a different rate than that of the hemoglobin contained in the
erythrocytes (Wells et al. 1986).
During fall, males from Mud Patch and New York Bight
showed elevated hemoglobin, possibly a response to anthropogenic factors.
Abnormally elevated hematocrit or hemoglobin may be related to hemoconcentration,
dehydration stress, or polycythemia (Wedemeyer and Yasutake 1977), or
to osmoregulatory dysfunction (Heath 1995). Also during fall, significantly
elevated MCHC was measured in males from Merrimack River, Mud Patch,
and New York Bight, and may indicate a greater amount of hemoglobin per
unit of red blood cells and possibly swollen erythrocytes. Windowpane
from a polluted harbor in Long Island Sound had significantly higher
hematocrit and hemoglobin than other less-impacted areas, suggesting
increased hematopoiesis (Dawson 1990). Larsson et al. (1980) also
saw increased hematocrit and hemoglobin in female European flounder exposed
to titanium dioxide industrial effluent in the field, and noted that
flounder may try to compensate for impaired oxygen uptake by a release
of erythrocytes from the spleen. Increased hematocrit was observed in
rainbow trout stressed by chlorine exposure (Zeitoun et al. 1977).
CONCLUSIONS
Seasonally-induced variability was observed in yellowtail
flounder blood constituent levels. This variability suggests that blood
chemistry values are best defined in terms of a physiological reference
range (Luskova 1998).
Seasonal patterns were not always consistent among
the study locations and may reflect natural variability among locations
(Bidwell and Heath 1993). This variability may result from inherent differences
in regional oceanographic conditions, such as temperature and salinity,
but may also be influenced by handling stress, and/or stock-related variations
or a combination of these factors (McCarthy et al. 1973; Hille
1982).
Our results present some evidence for possible anthropogenic
effects on certain blood chemistry parameters of yellowtail flounder
collected from inshore locations. Perhaps the most valuable aspect of
this study, though, is that it presents a benchmark for changes due to
environmental factors or anthropogenic influences over an annual cycle
at key locations in the Northeast U.S. Shelf Ecosystem.
ACKNOWLEDGMENTS
The authors thank Anthony Calabrese, Ronald Goldberg,
and Frederick Thurberg for editorial assistance, Suellen Fromm for providing
the NEMP hydrographic data, Christine Zetlin for designing Figure 1,
and Jose Pereira and Dee Tucker for technical assistance.
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Common and Scientific Names of Species Discussed |
| Atlantic halibut .......................................................................................................Hippoglossus
hippoglossus |
| Brook trout ......................................................................................................................Salvelinus
fontinalis |
| Channel catfish ..................................................................................................................Ictalurus
punctatus |
| English sole...........................................................................................................................
Parophrys vetula |
| Eurasian perch .......................................................................................................................
Perca fluviatilis |
| European flounder ...............................................................................................................
Platichthys flesus |
| Lake trout .................................................................................................................................
S. namaycush |
| Largescale blackfish...............................................................................................................
Girella punctata |
| Madai ........................................................................................................................................Pagrus
major |
| Mummichog ...................................................................................................................Fundulus
heteroclitus |
| Pinfish ...........................................................................................................................
Lagodon rhomboides |
| Plaice ...........................................................................................................................
Pleuronectes platessa |
| Rainbow trout ...............................................................................................................
Oncorhynchus mykiss |
| Redear sunfish ................................................................................................................Lepomis
microlophus |
| Rock bass......................................................................................................................
Ambloplites rupestris |
| Smallmouth bass ...........................................................................................................
Micropterus dolomieu |
| Striped bass ..........................................................................................................................
Morone saxatilis |
| Striped mullet ..........................................................................................................................
Mugil cephalus |
| Summer flounder ...........................................................................................................Paralichthys
dentatus |
| Windowpane ..............................................................................................................
Scophthalmus aquosus |
| Winter flounder .............................................................................................Pseudopleuronectes
americanus |
Acronyms |
| MCHC .........................................................................................
mean corpuscular hemoglobin concentration |
| NEFSC .....................................................................................................
Northeast Fisheries Science Center |
| NEMP...............................................................................................................
Northeast Monitoring Program |
| OPP ................................................................................................................................Ocean
Pulse Program |
|
